Bacteriorhodopsin, the chromoprotein of the purple membrane of halobacteria, acts as a light driven proton pump converting light energy into electrochemical potential. The stored electrochemical energy is used by the cell to drive the synthesis of ATP and other endergonic processes. We propose to refine characterization of the photoreaction cycle and the protonation changes in isotropic suspension of purple membranes and also the proton translocation in cell ghosts and in intact cell preparations. We will systematically study the light induced absorption changes in membrane suspensions by a combination of flash and low temperature spectroscopy under a variety of environmental conditions including pH, temperature, ionic strength and composition, light intensity, etc. Proton exchanges with the medium will be monitored spectroscopically with indicator dyes, stoichiometry and quantum yields will be calculated. We will also measure spectral changes in the ultraviolet to follow intramolecular protonation changes of suitable groups. Photoselection spectroscopy and photoinduced dichoisms will be used to study chromophore motions during the photo-reaction cycle. The coupling of the proton electrochemical gradient to energy requiring processes will be studied in cell ghosts and intact cell preparations. Simulataneous measurement of electric transmembrane potentials, proton and other ion gradients and ATP at about one second resolution will be used to establish kinetic correlations between these processes. We will measure these parameters by determining the distribution of physiologic and added permenant ions, weak acids and pH and membrane potential indicator dyes, using spectroscopic techniques and radioactive tracers.